Method of producing succinic acid and other chemiclas using facilitated diffusion for sugar import

ABSTRACT

This invention relates to the production of succinic acid and other chemicals derived from phosphoenolpyruvate (PEP) by fermentation with a microorganism in which the fermentation medium contains one or more sugars, and in which one or more of the sugars is imported into the cell by facilitated diffusion. As a specific example, succinic acid is produced from a glucose-containing renewable feedstock through fermentation using a biocatalyst. Examples of such a biocatalyst include microorganisms that have been enhanced in their ability to utilize glucose as a carbon and energy source. The biocatalysts of the present invention are derived from the genetic manipulation of parental strains that were originally constructed with the goal to produce one or more chemicals (for example succinic acid and/or a salt of succinic acid) at a commercial scale using feedstocks that include, for example, glucose, fructose, or sucrose. The genetic manipulations of the present invention involve the introduction of exogenous genes involved in the transport and metabolism of glucose or fructose into the parental strains. The genes involved in the transport and metabolism of glucose or fructose can also be introduced into a microorganism prior to developing the organism to produce a particular chemical. The genes involved in the transport and metabolism of sucrose can also be used to augment or improve the efficiency of sugar transport and metabolism by strains already known to have some ability for glucose utilization in biological fermentations.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the priority of the U.S. Provisional ApplicationSer. No. 61/857,300, filed on Jul. 23, 2013.

FIELD OF THE INVENTION

The present invention is in the field of producing specialty andcommodity organic chemicals using biocatalysts (bacteria and othermicroorganisms) that can be modified to increase their efficiency inusing sugar-containing feedstocks. More specifically, the presentinvention is related to the genetic modifications of genes that encodefunctions involving transport and metabolism of sugars for thebiological production of succinic acid and other chemicals.

BACKGROUND OF THE INVENTION

A large number of organic chemicals are currently derived frompetrochemical feedstocks. There is a growing interest in producing manyof these petrochemical-derived organic compounds through biologicalfermentation processes using renewable feedstocks. The list of organiccompounds that can be derived from renewable feedstocks includesα,ω-diacids (succinic, fumaric, malic, glucaric, malonic, and maleic),2,5-furan dicarboxylic acid, propionic acid, 3-hydroxy propionic acid,aspartic acid, glucaric acid, glutamic acid, itaconic acid, levulinicacid, 3-hydroxybutyrolactone, glycerol, and butanediols such as 1,4butanediol (US Patent Application 20090047719), 1,3-butanediol (USPatent Application 20090253192), and 2,3-butanediol. Many other types oforganic compounds, including, but not limited to, amino acids, vitamins,alcohols (such as ethanol, n-propanol, isopropanol, n-butanol,isobutanol, and higher alcohols), fatty acids, esters of fatty acids,hydrocarbons, isoprenoids, turpenes, carotenoids, amines, can also beproduced using renewable feedstocks. Any such compound shall be referredto herein as a “desired compound”. Although fermentation processes formany of these desired compounds have been developed, in order to competewith petrochemical processes, there is a constant need to improve theoverall economics of fermentation, for example to improve product titer(final concentration in grams per liter of product) and product yield(grams of product per gram of carbon source such as glucose), and toreduce the titer of unwanted byproducts, such as acetate.

Many bacteria, including Escherichia coli, use a system for activelytransporting glucose and other sugars into the cell called aphosphotransferase system (PTS). This system uses PEP (phosphoenolpyruvate) as the source of energy and phosphate for simultaneouslytransporting and phophorylating the sugar. PTS systems usually requirefour or more proteins that together function to import and phosphorylatethe incoming sugar. Some of these proteins are common to all of thesugars that a given organism imports by a PTS, while other proteincomponents of the PTS are specific for one or more particular sugars.

For example, in E. coli, the proteins that are common to all PTSpathways are PtsH and PtsI, encoded by the genes ptsH and ptsI,respectively. In addition to these two “common” PTS proteins, one ormore additional sugar-specific PTS proteins are required to import andphosphorylate particular sugars. For example, import of glucose by thePTS requires two additional proteins named Crr and PtsG. Crr is acytoplasmic protein with a single domain called A, and PtsG is amembrane protein with two domains named B and C. The phosphate groupfrom PEP is relayed from protein to protein and is finally transferredto glucose as it is imported, at the 6 position to giveglucose-6-phosphate inside the cell. The order of the relay startingwith PEP is PtsI, PtsH, Crr, and finally PtsG. Historically, theseproteins have also been called by other names, such as EI, HPr,EIIA^(Glc), and EIIBC, respectively. As another example from E. coli,fructose is imported by a similar relay using PtsI, PtsH, FruA, andFruB, the last two of which are also known as EII^(Fru) and EII^(Fru),respectively. For some sugars, for example mannitol, the sugar-specificprotein domains corresponding to A, B, and C as mentioned above forglucose are fused into one membrane bound polypeptide, while for othersugars, for example mannose, the A and B domains are fused into onecytoplasmic polypeptide, while the membrane bound component is comprisedof two subunits called C and D.

In all cases, the system relies on the “common subunits” (PtsI and PtsHin E. coli), and PEP is the source of energy and phosphate. As a result,every molecule of sugar imported by a PTS system results in theutilization of one molecule of PEP and the production of one molecule ofphosphorylated sugar and one molecule of pyruvate. However, PEP is alsoan obligate intermediate in several biochemical pathways, such as 1)formation of pyruvate and ATP by pyruvate kinase, 2) the anapleuroticpathways catalyzed by PEP carboxykinase and PEP carboxylase, which bothfeed carbon into the TCA (tricarboxylic acid) cycle, and 3) the entryinto the common aromatic amino acid and aromatic vitamin biosyntheticpathway catalyzed by one or more isozymes of3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (DAHP synthase).Thus there is an inevitable competition for PEP between the PTS systemfor sugar import and the other pathways just mentioned.

Since many bacteria, including both Gram positives and Gram negatives,use a PTS system, it is obviously a system that has prevailed under manycircumstances throughout evolution. However, under anaerobic conditions,production of ATP from sugars such as glucose is much less efficientthan under aerobic conditions, and the so-called “substrate level”phosphorylation, for example, by pyruvate kinase, becomes a largerportion of the ATP production budget than under aerobic conditions whereoxidative phosphorylation provides the majority of the ATP budget. Assuch, it is noteworthy that some organisms, such as Saccharomycescerevisiae and Zymomonas mobilis, both of which are well adapted toanaerobic growth on glucose and other sugars, do not have a PTS system,but instead use a facilitated diffusion protein (also called a permease)to import glucose and other sugars. Furthermore, when organisms thatnatively use a PTS are genetically engineered to overproduce particularcompounds by fermentation, the pathways in many cases use PEP as anintermediate, so that the PTS competes with the desired biosyntheticpathway for PEP. Alleviation from this competition by reducing theactivity of the PTS is known to increase flux to a desired biosyntheticpathway.

For example, PEP is an intermediate in the reductive branch of thetricarboxylic acid (TCA) cycle that leads to succinate. During themetabolic evolution of KJ122, an E. coli succinate producer, aframeshift mutation arose in the ptsI gene, which resulted in anincrease in succinate production from glucose. Reinstalling a wild typeptsI gene caused a drastic reduction in succinate production, provingthat the ptsI mutation contributed strongly to the strain improvement.

For another example, aromatic amino acids are built from PEP anderythrose-4-phosphate. Deletion of three pts genes (ΔptsHI, crr) in anE. coli strain was shown to increase flux to the aromatic amino acidbiosynthetic pathway when cells are grown on glucose as the carbonsource.

In both of the above examples, import of glucose is presumably stillaccomplished at some level by the so-called galactose permease (GalP,encoded by the galP gene). In the first example, a mutation that reducedthe activity of a repressor (GalS) of the galP gene was found to resultfrom metabolic evolution (WO2011/123154). In the second example, one ormore mutations occurred after deletion of pts genes that resulted in anincrease in growth rate. The resulting strain depended on galP forsignificant growth on glucose, and one or more mutations in the straincould have been related to an increase in expression of galP (U.S. Pat.No. 6,962,794). However, the strains from this second example producedonly low titers of aromatic amino acids after engineering the“Pts−/Glu+” strains for aromatic amino acid production. Phenylalanine,tyrosine, and tryptophan were produced at 1.7, 0.8, and 2.2 g/lrespectively. Since these titers are nowhere near high enough to supportan economically attractive commercial process, it is not clear that theinvention disclosed in U.S. Pat. No. 6,962,794 is useful for commercialproduction. As such, there is still a need to improve fermentationparameters for economically viable commercial production of chemicals byfermentation.

Although the use of GalP for glucose import conserves PEP, it is aproton symporter, so it consumes about ⅓ of an ATP for each glucosemolecule transported. Some microorganisms, for example the bacteriumZymomonas mobilis and the yeast Saccharomyces cerevisiae use facilitateddiffusion for importing glucose. Z. mobilis has one facilitator proteinthat functions to import both glucose and fructose. S. cerevisiae has atleast 14 different hexose importers, many of which import glucose and atleast some of which import fructose as well. This mode for glucoseimport requires no ATP expenditure until the sugar is inside thecytoplasm, after which an ATP is consumed to form glucose-6-phophate toallow the sugar to enter glycolysis. Most importantly, unlike for thePTS system, no PEP is consumed. As such, facilitated diffusion clearlyworks well for some organisms, and costs the cell less in terms of PEPand ATP than either a PTS system or a proton symporter such as GalP.Ingram et al. (U.S. Pat. No. 5,602,030) demonstrated that thefacilitated diffusion protein (Glf, encoded by the glf gene) fromZymomonas mobilis, together with a glucokinase (Glk, encoded by the glkgene), also from Zymomonas mobilis, expressed from those genes on amulticopy plasmid, could functionally replace the PTS to support growthin a minimal glucose medium of an E. coli strain, where the parent hadno native glucose facilitated diffusion capability, and other glucoseimport systems had been disabled by mutation. The recombinant E. coliptsG−, ptsM−, glk− strain ZSC113 containing the two Z. mobilis genes glfand glk on a plasmid could grow aerobically on minimal glucose medium.

These disclosures proved that the Z. mobilis proteins could function inE. coli enough to support growth aerobically with a specific growth rateof 0.53 hr-1. However, wild type E. coli using the native PTS forglucose import has an aerobic specific growth rate of 1.0 to 1.2 hr-1),so the strains engineered in U.S. Pat. No. 5,602,030 to use glf appearto be severely limited by glucose uptake. Moreover, the disclosures didnot show that the facilitated diffusion system could support anaerobicgrowth. A number of important chemicals produced by fermentation requirerobust anaerobic growth to support an economically attractive commercialproduction system (WO2012/018699). The examples in U.S. Pat. No.5,602,030 and Snoep et al (1994) showed that modest growth could beobtained by expressing glf and glk from a multicopy plasmid, but it wasnot demonstrated that growth could be supported by integrated copies ofthe glf and glk genes, yet it is often desirable for commercial scaleproduction to use strains that do not contain a plasmid. Finally, U.S.Pat. No. 5,602,030 did not demonstrate that a glf-based system couldsupport high titer production of a commodity chemical such as ethanol orsuccinate in E. coli or any other organism that does not natively usefacilitated diffusion. As such, it was not clear from the disclosure ofU.S. Pat. No. 5,602,030 alone that a glf could replace the PTS andresult in an economically attractive fermentation processes forproducing a desired chemical in a host strain that does not have anative facilitated diffusion system.

Tang et al (2013) went a couple steps further to show that anaerobicproduction of succinate could be achieved by expression of Z. mobilisglf in combination with a glucokinase in an E. coli strain backgroundthat was ΔptsI, ΔldhA, ΔpflB, pck*. However, the best succinateproduction in this system was modest, only 220 mM (26 g/l) in 96 hours.Despite having optimized by combinatorial modulation the expression ofglf and glk, this titer and productivity is nowhere near that ofpreviously published strains that produced 83 g/l succinate without theuse of glf. Thus, despite the more advanced work of Tang et al., it hadstill not been demonstrated that the use of facilitated diffusion forglucose import was useful for actually improving fermentation productionparameters at levels that would be necessary for economically attractivecommercial production, which would be at the benchmark of at least 83g/l (WO2012/018699). To further complicate the potential replacement ofa PTS by glf, in E. coli, and presumably in other bacteria, thecomponents of the PTS have many diverse regulatory functions that affectmany different metabolic pathways, so it is impossible to predict whatthe effects will be of a deletion in any one or more of the PTS genes onthe overall physiology and fermentative properties of any resultingmodified strain. Native Z. mobilis strains, which naturally usefacilitated diffusion for glucose uptake, are capable of producing up toabout 60 g/l ethanol and a similar quantity of carbon dioxide fromglucose. An engineered strain of Z. mobilis is reported to produce 64g/l succinate from glucose (EP20070715351). However, this fermentationrequired 10 of yeast extract in the fermentation medium, which isundesirable for commercial production of succinic acid, both because ofits expense and the increased cost required for downstream purificationof the succinate from the yeast extract components. Furthermore, Z.mobilis is often not a convenient or optimal host organism for use infermentative processes.

Thus, to summarize the prior art, it had been shown that E. coli can beengineered to use facilitated diffusion of glucose to support aerobicgrowth to a modest rate, and to support a modest level of succinateproduction anaerobically, but there has been no disclosure of anybacterial strain or process that has been engineered to confer thenon-native use of facilitated diffusion for glucose import and that isimproved over strains using native glucose import systems such as PTSand/or GalP for production of a chemical by fermentation. Furthermore,there has been no disclosure of any bacterial strain or process thatuses facilitated diffusion for glucose import and that is capable ofproducing succinate or any chemical other than ethanol and carbondioxide at a titer, yield, and rate that is high enough in a medium thatwould be commercially attractive, such as a minimal glucose medium. Assuch, there is still a need for improved strains that can producesuccinate and chemicals in a process that is economically attractivewhen all factors including productivity, cost of the medium, anddownstream purification are taken into account.

SUMMARY OF THE INVENTION

This present invention provides biocatalysts (for example geneticallyengineered microorganisms) and methods for using facilitated diffusionof glucose for improving the fermentative production of commerciallyimportant products, for example, but not limited to, specialty andcommodity chemicals. Specifically, the present invention is useful inthe fermentative production of organic acids, amino acids, and otherbiochemicals that have PEP as a biochemical intermediate in theirbiosynthetic pathway, using sugar-containing renewable feedstocks. As aspecific example, the present invention is useful in the fermentativeproduction of succinic acid from a glucose, fructose, orsucrose-containing renewable feedstock using biocatalysts that have beenconstructed to use facilitated diffusion of a sugar. The principles ofthe present invention can be applied to many other desired chemicalcompounds that can be produced by fermentation, particularly chemicalsintermediates of the TCA cycle or derivatives thereof, such as fumaricacid, malic acid, glutamate, derivatives of glutamate, aspartate,derivatives of aspartate, aromatic amino acids (phenylalanine, tyrosine,tryptophan), and compounds derived from intermediates in the centralaromatic pathway, such as vitamins and cis, cis-muconic acid.

According to the present invention, genes coding for the proteinsinvolved in facilitated diffusion of sugars such as glucose can beintroduced into a wide variety of biocatalysts either to confer a newability to the biocatalyst to import a sugar as a source of carbon andenergy from the fermentation medium by facilitated diffusion, or toaugment or improve an already existing capacity of the biocatalysts forsugar transport and metabolism. Strains engineered to have the addedability to import sugars by facilitated diffusion can have improvedfermentation parameters when compared to parameters of the parentstrain, such as increased titer (g/l of desired chemical product),increased yield (grams of product produced per gram of sugar consumed),increased specific productivity (g/l-hr of product formation), and/ordecreased titer of unwanted byproducts such as acetate, pyruvate and/oramino acids. These improved parameters can result from conservation ofenergy (for example use of less ATP for formation of proton gradients todrive proton symporters such as GalP), conservation of PEP for pathwaysthat use PEP as an intermediate, such as the succinate pathway(s), anddecreasing of overflow metabolism into acetate production pathways orother unwanted pathways.

This approach is particularly advantageous for production of chemicalsthat are derived at least in part from or through PEP, such assuccinate, malate, fumarate, lactate, ethanol, butanols, propane diols,3-hydroxypropionic acid, acrylic acid, propionic acid, lactic acid,amino acids such as glutamate, aspartate, methionine, lysine, threonine,and isoleucine, compounds derived from the central aromatic pathway suchas phenylalanine, tyrosine, tryptophan, aromatic vitamins, aromaticvitamin-like compounds, and any other compound that is derived from PEPas a biosynthetic intermediate.

In one embodiment, the present invention provides biocatalysts that donot natively have the ability to import a sugar by facilitated diffusionwith an added heterologous gene (or genes) that confers a new ability toimport a sugar by facilitated diffusion. In another embodiment, thepresent invention provides novel biocatalysts that produce a highertiter of a desired fermentation product than the parent biocatalyst. Inanother embodiment, the present invention provides novel biocatalyststhat produce a higher yield of a desired fermentation product than theparent biocatalyst. In another embodiment, the present inventionprovides novel biocatalysts that produce a higher specific productivityfor a desired fermentation product than the parent biocatalyst. Inanother embodiment, the present invention provides a novel biocatalystthat produces a lower titer of an undesired desired byproduct than theparent biocatalyst.

The gene or genes that code for the protein or proteins that function inthe facilitated diffusion of a sugar can be derived from any organismthat has the native ability to carry out facilitated diffusion of asugar, the only requirement being that the protein or proteins are ableto function in the new host. The gene encoding a sugar kinase, forexample a glucokinase, that is required to phosphorylate the sugar afterit enters the cytoplasm can be derived from the same donor from whichcame the gene(s) for facilitated diffusion, or a native sugar kinasegene from the recipient host can be used, or a combination of both sugarkinases can be used.

In another embodiment, the present invention provides for methods forproducing a desired fermentation product comprising cultivating agenetically engineered microorganism that used facilitated diffusion toimport a sugar.

In another embodiment, the present invention provides for methods forimproving fermentation performance parameters (titer, yield, specificproductivity, minimizing byproduct formation) of strains engineered touse facilitated diffusion.

In another embodiment, the present invention provides for methods forachieving an improved balance of facilitated diffusion and sugar kinaseactivity leading to improved growth and fermentation parameters ingenetically engineered microorganism that used facilitated diffusion toimport a sugar.

According to the present invention, one approach is to geneticallytransfer a facilitated diffusion system for importing a sugar from asecond donor organism that naturally contains the relevant genes (forexample glf or glk or a combination thereof) into a first recipientorganism that does not naturally contain said relevant genes, so as toconfer on said first recipient organism a new ability to import saidsugar by facilitated diffusion. In a preferred embodiment, the firstrecipient has been previously engineered or constructed to be devoid of,or substantially reduced in, its ability to import said sugar by anynative system or systems present in a parent or ancestor of said firstrecipient strain. In such an embodiment, the resulting strain is ineffect forced to use facilitated diffusion for growth on said sugar.

In a preferred embodiment, the first recipient strain is an E. colistrain, and the second donor strain is Zymomonas mobilis CP4. In a morepreferred embodiment, said first strain is WG53, which in turn isderived from KJ122 by deletion of ptsH, ptsI, and galP. The exact natureof the deletions of ptsH, ptsI, and galP can vary widely, the onlyimportant criterion being that the activities of the PtsH, PtsI, andGalP proteins are eliminated or substantially reduced.

The first recipient organism of the invention can vary widely, the onlycriterion being that it does not natively contain a protein thatfunctions in facilitated diffusion for a sugar such as glucose. Inaddition to E. coli, examples of first recipient organisms include, butare not limited to: Gluconobacter oxydans, Gluconobacter asaii,Achromobacter delmarvae, Achromobacter viscosus, Achromobacter lacticum,Agrobacterium tumefaciens, Agrobacterium radiobacter, Alcaligenesfaecalis, Arthrobacter citreus, Arthrobacter tumescens, Arthrobacterparaffineus, Arthrobacter hydrocarboglutamicus, Arthrobacter oxydans,Aureobacterium saperdae, Azotobacter indicus, Brevibacteriumammoniagenes, divaricatum, Brevibacterium lactofermentum, Brevibacteriumflavum, Brevibacterium globosum, Brevibacterium fuscum, Brevibacteriumketoglutamicum, Brevibacterium helcolum, Brevibacterium pusillum,Brevibacterium testaceum, Brevibacterium roseum, Brevibacteriumimmariophilium, Brevibacterium linens, Brevibacterium protopharmiae,Corynebacterium acetophilum, Corynebacterium glutamicum, Corynebacteriumcallunae, Corynebacterium acetoacidophilum, Corynebacteriumacetoglutamicum, Enterobacter aerogenes, Erwinia amylovora, Erwiniacarotovora, Erwinia herbicola, Erwinia chrysanthemi, Flavobacteriumperegrinum, Flavobacterium fucatum, Flavobacterium aurantinum,Flavobacterium rhenanum, Flavobacterium sewanense, Flavobacterium breve,Flavobacterium meningosepticum, Micrococcus sp. CCM825, Morganellamorganii, Nocardia opaca, Nocardia rugosa, Planococcus eucinatus,Proteus rettgeri, Propionibacterium shermanii, Pseudomonas synxantha,Pseudomonas azotoformans, Pseudomonas fluorescens, Pseudomonas ovalis,Pseudomonas stutzeri, Pseudomonas acidovolans, Pseudomonas mucidolens,Pseudomonas testosteroni, Pseudomonas aeruginosa, Rhodococcuserythropolis, Rhodococcus rhodochrous, Rhodococcus sp. ATCC 15592,Rhodococcus sp. ATCC 19070, Sporosarcina ureae, Staphylococcus aureus,Vibrio metschnikovii, Vibrio tyrogenes, Actinomadura madurae,Actinomyces violaceochromogenes, Kitasatosporia parulosa, Streptomycescoelicolor, Streptomyces flavelus, Streptomyces griseolus, Streptomyceslividans, Streptomyces olivaceus, Streptomyces tanashiensis,Streptomyces virginiae, Streptomyces antibioticus, Streptomyces cacaoi,Streptomyces lavendulae, Streptomyces viridochromogenes, Aeromonassalmonicida, Bacillus pumilus, Bacillus circulans, Bacillusthiaminolyticus, Escherichia freundii, Microbacterium ammoniaphilum,Serratia marcescens, Salmonella typhimurium, Salmonella schottmulleri,Bacillus subtilis, Bacillus licheniformis, Bacillus amylolliquefaciens,Klebsiella oxytoca, Klebsiella pneumoniae, Acinetobacter baylyi,Corynebacterium glutamicum Brevibacteium flavum, Mannhemiasucciniproducens and Anaerobiospirilum succiniproducens, and Xanthomonascitri.

Examples of second donor organisms are any strain or species that has anative facilitated diffusion system for a sugar, for example Zymomonasmobilis strains (in addition to strain CP4), Homo sapiens, Azospirillumamazonense, Flavobacteriaceae bacterium S85, Saccharomyces cerevisiae orother yeast genera.

In another embodiment, a first parent strain is first constructed to usefacilitated diffusion for importing a sugar, and then the resultingstain is further engineered to overproduce a chemical of commercialinterest such as succinic acid.

Novel aspects of this invention are that the glf gene from anon-pathogenic, robust sugar utilizer has been stably integrated intothe chromosome of a bacterium, such that the newly constructed bacteriumcan produce a commercially viable product with an economically viableprocess. The titer, yield and/or specific productivity of product fromglucose or another sugar is greater than those parameters of the parentorganism. The glf gene is integrated at a site in the chromosome thatdoes not interfere with any relevant aspect of growth or productproduction. The acetate titer is less than that of the parent strain atabout 45 to 48 hours in a representative fermentation, allowing a 2 dayfermentation cycle time, unlike a prior art example. Strains in theprior art that used facilitated diffusion for sugar import did notproduce sufficient titers of the desired product to be economicallyattractive. Another novel aspect of this invention is that by usingfacilitated diffusion for sugar import, it was unexpectedly found thatthe production of the unwanted byproduct acetate or acetic acid wassignificantly reduced. The prior art strain KJ122 produces about 5 to 7g/l acetate in a typical fed glucose fermentation (WO2012/018699), whilenew strains of the invention produce only about 4.2 g/l or less.

Additional advantages of this invention will become readily apparentfrom the ensuing description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Structure of plasmid pAC19, a source of an expression cassettefor Z. mobilis glf and glk.

FIG. 2 Structure of plasmid pAC21, a source of a selectable andcounter-selectable cassette containing cat (chloramphenicol resistance)and sacB (levan sucrase) genes.

FIG. 3 Structure of plasmid pSS2, a source of an expression cassette forZ. mobilis glf without glk.

FIG. 4 Structure of plasmid pMH68, a source of an expression cassettefor integration of a second copy of the E. coli crr gene at the pflDlocus.

Table 1. Production of succinate by AC15 in 7 liter fermentors

Table 2. Production of succinate by red mutants of AC15 in 500 mlmicroaerobic fermentors.

Table 3. Production of succinate by two isolates of SS8 in 500 mlmicroaerobic fermentors.

Table 4. Production of succinate by YSS41 in 20 liter microaerobicfermentors.

Table 5. Production of succinate by MH141 in 500 ml microaerobicfermentors.

Table 6. Succinate production by E. coli strains KJ122 and YSS41 in 20liter fermentors under optimized aeration conditions for both strains.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Definitions

When the phrase “for example” or “such as” is used, the subsequentlymentioned items are meant to be illustrative examples for the idea orconcept being disclosed. The subsequently mentioned items are not meantto be limited to the examples given, since any other specific item orexample that would fall under the generalization of the idea or conceptis meant to be included. For any given compound, it might be moreappropriate to produce a salt of said compound, so for example, succinicacid might be produced at pH near 7 as a salt of sodium, potassium,calcium, magnesium, ammonium, etc., while lysine might be produced as asalt of chloride, sulfate, bicarbonate, etc. As such, any time acompound is named herein, any salt of said compound is meant to beincluded, and any time a salt is named, the free acid or free base isalso meant to be included. Thus, for example, “succinate” is meant toinclude “succinic acid” and vice versa, and “acetate” is meant toinclude “acetic acid” and vice versa.

“Facilitated diffusion” means the action of a system, typicallycomprising an integral membrane protein situated in a biologicalmembrane (for example the inner membrane of a Gram negative bacterium orthe single membrane of a Gram positive bacterium), or a complex of morethan one protein molecules situated in a biological membrane, thatfunctions to specifically allow one or more chemicals called the“substrate” (for example glucose and/or fructose), but not chemicals ingeneral (for example water and cytoplasmic metabolites other than thespecific substrate), to cross through the membrane without any energy(such as that provided by hydrolysis of ATP or PEP) or gradient of adifferent chemical (for example a proton gradient) provided directly tothe system by the cell. If there is a concentration gradient, forexample if the concentration of a substrate is higher outside the cellthan inside the cell, there will be a net flux of that substrate intothe cell at a rate that is faster than would occur if the facilitateddiffusion system were absent. The protein(s) that function forfacilitated diffusion typically have a binding affinity that is specificfor one or more substrates and allows the system to assist passing thesubstrate across the membrane at relatively low concentrations ofseveral millimolar or less. Some types of facilitated diffusion canfunction by creating a pore or channel through the membrane thatdiscriminates in favor of a substrate, and in other types the protein(s)can bind the substrate on one side of the membrane and then rotatethrough the membrane to release the substrate on the opposite side ofthe membrane. A facilitated diffusion protein (sometimes called simply afacilitator) is a protein component of such a system. Thus, thethermodynamic driving force for facilitated diffusion is a gradient ofsubstrate concentration, in which the substrate (for example a sugar)flows from a higher concentration outside of a cell to a lowerconcentration inside the cell. We shall use the genetic symbols Glf andglf to respectively mean a facilitated diffusion protein and a geneencoding such a protein that has specificity for glucose. We usuallyconsider Glf to be a comprised of a single polypeptide chain, but a Glfcould be a complex comprised of more than one polypeptide chain.Although the specific examples of Glf written herein are bacterial inorigin, our definition is meant to include facilitated diffusion systemderived from any organism. For example, it is well known that the yeastSaccharomyces cerevisiae and other yeasts have one or more facilitateddiffusion proteins for importing hexoses (for example glucose andfructose) named HXT1, HXT2, HTX3, HTX4, HTX5, HTX6, HTX7, etc.), andhuman erythrocytes use facilitated diffusion to import and exportglucose via a protein named GLUT1. The mechanism of action of Glf's canvary widely, including pore-facilitated transport andcarrier-facilitated transport. Although the specific examples given inthis specification disclose a Glf that has good specificity for glucose,it is known in the art that a Glf protein can be active on more than onesugar, for example Glf from Zymomonas mobilis and Saccharomycescerevisiae can be active on fructose as well as glucose.

Proton symport is defined as a system for importing a substrate across abiological membrane that uses a proton gradient as a driving force. Ahigher concentration of protons outside of the cell has a thermodynamictendency to diffuse back into the cell. This thermodynamic pressure isused to carry in a substrate such as a sugar. A proton symporter is aprotein or complex of proteins that functions to provide proton symport.An example of a proton symporter is the GalP protein of E. coli, whichis well known to function in the import of galactose, glucose, and othersugars.

A glucokinase and a fructokinase are enzymes that catalyzephosphorylation of glucose, fructose, or other sugar, usually at the6^(th) carbon position, but alternatively possibly at the 1^(st) carbonor another position. We shall use the genetic symbols Glk and glk torespectively mean a glucokinase and a gene that encodes a glucokinase.Frk and frk mean a fructokinase and a gene that encodes a fructokinase,respectively.

A crr gene is a gene that encodes an EIIA^(glc) component of a PTS, suchas the crr gene of an E. coli strain or of a Bacillus subtilis strain ora homolog of such a crr gene.

A PTS (phosphotransferase system) is a group of proteins that acttogether to pump a sugar into a cytoplasm and simultaneouslyphosphorylate the sugar, using PEP as the source of phosphate andenergy. Examples of genes encoding PTS proteins from E. coli includeptsH, ptsI, crr, ptsG, fruA, fruB, manX, manY, and manZ. Thecorresponding proteins are named PtsH, PtsI, Crr, PtsG, FruA, FruB,ManX, ManY, and ManZ. However, there are many more examples from E. coliand other prokaryotes, and these proteins can have alternate names, forexample Crr is sometimes named EIIA^(glc). Some of the PTS proteins aremore specific to one or more particular sugars than to other sugars,while some PTS proteins, for example PtsH and PtsI from E. coli, areused in common for many different sugars.

In this specification, the term “microaerobic” means that the feed rateof air is less than 0.1 volume of air per volume of liquid culture perminute. In 7 and 20 liter fermentor examples disclosed herein, this isaccomplished with a sparger and flow meter, or by allowing the tank tobreathe through a sterile membrane attached to the top of the tankwithout any forced air flow. In 500 ml fermentor tank examples disclosedherein, no air is deliberately introduced, but a small amount of air isintroduced from leakage, feeding of the base solution, and taking ofsamples.

A “minimal medium” is a microbial growth medium comprised of water, apure carbon source (such as a substantially pure sugar or mixture ofsubstantially pure sugars), mineral salts (for example potassium,sodium, magnesium, calcium, bicarbonate plus carbonate, phosphate,sulfate and chloride), a pure nitrogen source such as ammonium or urea,trace metals (iron, copper, zinc, manganese, cobalt, molybdenum, andoptionally borate), optionally glycine betaine (also known as simplybetaine), and optionally an antifoam agent. Minimal media do not containany complex (also known as “rich”) nutrient source such as yeastextract, corn steep liquor, soy hydrolysate, broth, casein hydrolysate,grain, legume, or any other “undefined” mixture of nutrients thattypically would be derived from an agricultural source without anyphysical or chemical purification or separation steps. Reasonably puresugars derived from sugar cane, corn starch, sorghum starch, tapiocastarch, or any other reasonably pure starch source is considered to beacceptable for a minimal medium. A minimal medium can contain one or afew pure chemicals to satisfy a particular growth requirement(auxotrophy or bradytrophy) or to enhance a biochemical pathway. Forexample, some strains require a vitamin such as biotin, which can beadded at small concentrations without a significant negative impact on aprocess. As another example, addition of a vitamin such as thiamine,while not absolutely required for growth, can nonetheless enhance growthor a biochemical pathway. Minimal media are preferable for fermentativeproduction of many chemicals due to the relatively low cost of thecomponents, and due producing cleaner fermentations broths that allowsfor more favorable economics for downstream purification of the desiredchemical. Ethanol production is an exception, since downstreampurification can be accomplished with distillation, an economicallyattractive method for purification of the desired product even fromcomplex media.

An aromatic biochemical means any one or more of the following:phenylalanine, tyrosine, or tryptophan, or any derivative thereof (suchas L-dihyroxyphenylalanine, melatonin, indole, indole acetic acid,indigo, serotonin, cinnamic acid, hydroxy styrene), a vitamin orvitamin-like compound containing an aromatic moiety (such asp-hydroxybenzoic acid, 2,3-dihyroxybenzoic acid, p-amino benzoic acid,folate, tocopherol, pyrroloquinoline quinone).

A homolog of a first gene or protein is defined as a second gene orprotein in which the second protein or the protein inferred to betranslated from the second gene has the same or a similar biochemicalfunction as the first protein or protein inferred to be translated fromthe first gene, and in which an alignment of the first and secondproteins or first and second inferred translated proteins results in a25% or greater identity or similarity for a region of at least 50 aminoacids in length, when using the default parameters of a publicallyavailable computer alignment program such as BLAST.

A mutation is any change in a DNA sequence relative to the DNA sequenceof the related wild type or native gene. A mutation can be a single ormultiple base change that introduces a premature stop codon or an aminoacid that is different from the wild type amino acid at that position. Amutation can be an insertion or deletion of one or more bases thatcreates a frame shift that results in a protein that is significantlydifferent from the wild type protein. A mutation can be a deletion thatremoves much, most, or all of a coding region (also known as an openreading frame or orf). One type of mutation removes one or more entireorfs plus additional non-coding DNA either upstream or downstream fromthe coding region, or both. A mutation can result from insertion of arelatively large DNA sequence (more than about 100 bases), for examplean insertion element (for example IS186 or IS4) or a transposon (forexample Tn10). When the intent is to remove a function, a preferablemutation is a deletion of all or most of an orf, however, smallermutations such as single base changes or insertions can often accomplishremoval of a function for all practical purposes. Mutations can bespontaneous, induced by mutagenesis, or constructed by geneticengineering. Some mutations, when desired to accomplish a strainimprovement, are mutations that decrease or eliminate a biologicalfunction, such as particular elements of a PTS. However, some mutations,when desired to accomplish a strain improvement, are mutations thatincrease a biological function, for example a “promoter up mutation” canincrease the expression of a desired gene, such as a glf gene.

“Exogenous” means a gene or protein derived from a second genus that hasbeen installed in a first genus, where said second genus is a differentgenus from said first genus.

A gene is defined as a region of a chromosome that encodes a protein orenzyme, and is meant to include both the open reading frame thatcorresponds to the protein or enzyme and any DNA sequences surroundingthe open reading frame that contribute to controlling the level or rateof production of the protein or enzyme, such as promoters, ribosomebinding sites, operators, regulatory protein binding sites, DNAcorresponding to 5′ untranslated mRNA leader sequences, terminators, andantiterminator sites. When two or more open reading frames thatcorrespond to protein coding DNA sequences are under the control of asingle promoter and a single terminator, the whole region encompassingthe promoter, open reading frames corresponding to protein coding DNAsequences and the terminator is referred as an operon. For example, whenthe exogenous genes glf and glk are under the control of a singlepromoter and a single terminator, it is referred as glf-glk operon.

The present invention provides biocatalysts for succinic acid productionin high titer, yield and productivity using a minimal medium with asugar as a carbon source. The term “yield” as defined in this inventionrefers to the number of grams of product (such as succinic acid)produced per gram of sugar (such as glucose or sucrose) consumed. Theterm “productivity” as defined in this present invention refers to thenumber of grams of product (such as succinic acid) produced per liter ofculture per hour. The term “titer” is defined as the concentration ofproduct (such as succinic acid) in the fermentation broth in grams perliter. The desirable yield for succinic acid is in the range of 0.8-1.2grams of succinic acid produced per gram of sugar consumed. Thedesirable productivity for succinic acid in this present invention is inthe range of 1 gram or more of succinic acid produced per liter perhour. The desirable titer of succinic acid is greater than 26 g/l, ormore preferably greater than 64 g/l, and most preferably greater than 83g/l in a fermentation time of 48 hours or less.

The bacterial growth rate is measured in terms of the rate of increasein the optical density at 550 or 600 nanometers of a liquid cultureresulting from the bacterial multiplication. The bacterial growth rateis also expressed in terms of time required for doubling of bacterialcells. In the bacterial cells suitable for the present invention, thebacterial cells are expected to have a doubling time of between 20minutes and 3 hours.

According to the present invention, the biocatalyst for succinic acidproduction can be developed in two different ways. Under the firstapproach, a wild type bacterial species is genetically manipulated and,optionally, evolved, to grow efficiently using facilitated diffusion forimport of glucose or other sugar. Once such a strain is constructed,subsequent genetic manipulations are carried out in the metabolicpathways to obtain a bacterial strain that produces succinic acid oranother desired chemical with high titer, yield and productivity, forexample, by following methods known in the art.

The patent applications published under Patent Cooperation Treaty withthe publication No. WO 2010/115067 and United States Patent ApplicationPublication No. US 20100184171 provide the details about the geneticengineering techniques useful in generating a strain of E. coli withimproved succinic acid production capacity. These two patentapplications are incorporated herein by reference.

Under the second approach, a bacterial strain already developed to havea commercially attractive yield and productivity for a chemical such assuccinic acid as described in the patent application publications US20100184171 and WO 2010/115067 is used as a parental strain. Furthergenetic manipulations, and optionally, evolution, are then carried outwith this strain to obtain a bacterial strain that has the ability touse facilitated diffusion to import glucose or another sugar to producesuccinic acid at a commercially attractive titer, yield, andproductivity.

As a specific example, this present invention discloses biocatalysts andmethods that have improved ability over that of the prior art to producesuccinic acid at high enough titer, yield and productivity while gainingthe new ability to import a sugar by facilitated diffusion. For example,the KJ122 strain of E. coli described by Jantama et al. can be selectedas the starting strain for the present invention. The KJ122 strain of E.coli is reported to have the ability to produce succinic acid in aminimal medium at high titer and productivity.

The KJ122 strain of E. coli was derived from the E. coli C strainthrough gene deletions and metabolic evolution as described in US PatentApplication Publication No. 20100184171 and in the International PatentApplication Publication No. WO 2010/115067. These two patent applicationpublication documents providing details about the genetic changes thatled to the development of the KJ122 strain of E. coli are incorporatedherein by reference. KJ122 does not have any substantial ability toimport glucose as a source of carbon by facilitated diffusion in theproduction of succinic acid. The absence of this function in KJ122 isattributable to the lack of a gene that encodes a Glf protein. Theinventors have discovered genetic approaches that enable KJ122 to moreefficiently use glucose as a source of carbohydrate while retaining orimproving its original ability to produce succinic acid at high titer,yield, and productivity in a minimal medium.

The term “carbohydrate” as used in this invention includesmono-saccharides such as glucose, fructose, xylose, and arabinose,disaccharides such as sucrose, melibiose, maltose and lactose,trisaccharides such as raffinose and maltotriose, and higheroligosaccharides, and hydrolysates derived from the enzymatic orchemical digestion of polysaccharides such as starch, cellulose, andbiomass. Simple carbohydrates, those with from one to three saccharideunits, are referred to herein as “sugars”, for example glucose,fructose, sucrose, maltose, etc.

The terms “PTS⁺ organism” or “PTS⁺ bacterium” refers to a bacteriumwhich has the capacity for a carbohydrate transport based on a PTS. Theterm “non-PTS organism,” or “non-PTS bacterium” or “PTS⁻” bacteriumrefers to bacterial cells that are mutated in one or more genes thatencode a PTS function, such that the activity of the PTS is decreasedrelative to that of the wild type PTS.

In one aspect, the present invention discloses the addition of genes toan organism in order to install or increase the activity of one or moreproteins and/or enzymes involved in the import and conversion of a sugarinto metabolic intermediates such as glucose 6-phosphate, glucose1-phosphate, fructose 6-phosphate, or fructose 1-phosphate that can befurther metabolized by the cell. The genes that encode relevant proteinsor enzymes are chosen from a group consisting of a glf gene, an HXTgene, a glk gene, and a frk gene.

In another embodiment, the present invention provides a process forproducing succinic acid or another chemical using facilitated diffusionto import a sugar such as glucose as a renewable feedstock. In oneaspect, the present invention provides a process for producing succinicacid from a sugar-containing medium that makes use of a biocatalyst thathas a decreased activity in at least one protein of the organism'snative PTS system relative to that of the ancestral or parental strain.In another aspect, the present invention provides a process forproducing succinic acid or other chemical in a sugar-containing mediumthat makes use of a biocatalyst that has a decreased activity in atleast one protein of the organism's native sugar import system relativeto that of the ancestral or parental strain involving use of a proteinsymport system, such as GalP.

The present invention provides ways to manipulate a PTS and in turn thebacterial carbohydrate uptake system. Since EI and HPr proteins functionas “general” or “common” components of the PTS system, inactivation ofeither the ptsI gene coding for EI protein or the ptsH gene coding forHPr protein would lead to the complete inactivation of a PTS. There willbe substantially less carbohydrate transport through the PTS system inbacterial cells where the activity of ptsH or ptsI or both has beendecreased or eliminated. When the PTS is partially or completelyinactivated, the bacterial cell has to depend on one or more otheralternative permease systems for carbohydrate transport.

When there is active glucose transport through PTS, the EIIA^(Glc)remains unphosphorylated as there is a carbohydrate substrate foraccepting its phosphate group. However, when there is no glucose in themedium, the phosphorylated form of EIIA^(Glc) cannot transfer itsphosphate group to glucose and therefore it remains in itsphosphorylated state. The unphosphorylated EIIA^(Glc) mediates thephenomenon generally known as carbon catabolite repression (CCR). UnderCCR, when glucose is present in the growth medium, the transport and/orutilization of other carbohydrates in the medium is prevented until theglucose in the medium is decreased to a low concentration. The carboncatabolite repression results from the inhibitory effect ofunphosphorylated EIIA^(Glc) on permease systems or other systems ofcarbon source utilization. A number of permeases involved in thecarbohydrate transport are known to be inhibited by unphosphorylatedEIIA^(Glc), for example, LacY or lactose permease. In addition, theunphosphorylated EIIA^(Glc) is known to have a negative effect on thetranscription of number of genes involved in carbohydrate transport andmetabolism through its influence on the adenylate cyclase system.

Strain KJ122, good succinate producer, contains a frameshift mutation inthe ptsI gene, and this mutation is important for good succinateproduction. Thus it was surprising in the context of the currentinvention that further improvements in succinate production could bemade by deleting ptsHI and galP, and then installing a facilitateddiffusion system.

In another embodiment, the present invention provides a non-naturallyoccurring duplication of the crr gene that encodes the EIIA^(Glc)protein. The inventors discovered that strains containing a ptsHIdeletion, a galP mutation, and an installation of a functional glf gene,have an unexpected tendency to acquire a mutation in the crr gene whichcauses a decrease or elimination in function of the EIIA^(Glc) protein,which in turn causes an unexpected undesirable decrease in succinateproduction parameters. Duplication of the crr gene by integrating asecond copy of crr at a locus separate from the native crr locus solvesthis problem by greatly reducing the frequency of mutants that becomephenotypically crr negative.

The present invention will be explained in detail below. An examplebacterium belonging to the genus Escherichia of the present invention isa strain which is constructed from a parental strain that is notinitially capable of using facilitated diffusion for sugar import, butwhich after genetic engineering as disclosed herein harbors a glf gene,and optionally an exogenous glk gene, and has the ability to usefacilitated diffusion for import of glucose and fructose.

The exogenous genes introduced into the cell can be maintained withinthe cell on a self-replicating plasmid. A plasmid can be maintainedthrough antibiotic selection or complementation of a chromosomalmutation. However, when the exogenous genes are maintained within thebiocatalyst on a self-replicating plasmid within the cell, it isnecessary to assure the there is no unnecessary waste of energy andmaterials leading to the inhibition of growth, and a decrease in theyield or productivity of the organic material being manufactured usingthe biocatalyst. Preferentially, the exogenous genes are integrated intothe host chromosome so that there is no need to add any antibiotics tomaintain the plasmids within the cell, and little or no metabolic burdenis placed on the cell for plasmid maintenance. There are many possiblelocations within the cell for the integration of the exogenous genes.The preferential locations for integrating the exogenous genes withinthe E. coli chromosomal DNA include regions that do not encode anessential function for growth and product formation under commercialfermentation conditions.

When the exogenous genes are obtained as an operon, it is preferable toremove any possible negative regulatory genes or proteins from theoperon. It is ideal to have only the genes and proteins that functionpositively in facilitated diffusion and metabolism. Thus, expression ofa facilitated diffusion gene is preferably not inhibited by a repressoror by carbon catabolite repression.

The following examples are provided as a way of illustrating the presentinvention and not as a limitation.

Any bacterium that does not natively use a facilitated diffusion systemfor sugar import can be improved according to the present invention.

A bacterium of the present invention may be obtained by introduction ofone or more genes that enables utilization of facilitated diffusion intoa succinic acid producing strain such as KJ122 or other strainpreviously engineered to produce a desired chemical. Alternatively abacterium of the present invention may be obtained by conferring anability to produce succinic acid or other desired chemical to abacterium in which utilization of facilitated diffusion has already beenenabled by genetic engineering, and optionally by evolution. This latteralternative can be accomplished, for example, by following all the stepsused for constructing KJ122 but starting with strain ATCC 9637 or a K-12type E. coli strain, or any other safe E. coli strain, instead ofstarting with strain ATCC 8739.

Example 1 Construction of AC15, a Derivative of KJ122 that Contains theGlf and Glk Genes from Gene Cluster from Zymomonas mobilis CP4

All manipulations of DNA and plasmids, polymerase chain reaction (PCR),transformation, and chromosomal integration were accomplished bystandard methods that are well known in the art, It is well known thatDNA sequences can be cloned and joined together to form new combinationsthat cannot be easily found in nature. In addition to the moretraditional methods involving restriction enzymes and DNA ligase, newermethods using recombineering in yeast, the so-called “Gibson Method” ofin vitro splicing of DNA, or any other appropriate method can be used toconstruct such novel DNA sequences. The DNA fragments needed can beobtained from libraries of clones or by PCR from appropriate templateDNA. It is also understood that many desired DNA sequences can bedesigned and synthesized from chemical precursors. Such a service issupplied by a number of commercial companies, for example DNA 2.0 andGeneArt (Invitrogen).

Plasmid pAC19 was constructed to contain an artificial operon containingthe glf and glk genes from Z. mobilis, driven by the P₂₆ promoter fromthe Bacillus subtilis phage SP01. This operon was embedded between anupstream sequence homologous to the E. coli C tdcC gene and a downstreamsequence homologous to the E. coli C tdcE gene, to foster integrationinto the tdcCDE locus of strains to be engineered. The cassettedescribed above is carried on a low copy plasmid vector derived frompCL1921, which contains the pSC101 origin of replication and aspectinomycin resistance gene. The components for the cassette wereobtained by PCR using appropriate synthetic DNA primers obtained fromcommercial suppliers such as Sigma and Integrated DNA Technologies(IDT). The source for the Zymomonas genes was pLOI1740, which originallycontained a zwf and edd gene in addition to the desired glf and glkgenes. The glf, zwf, edd, glk cluster was transferred to pCL1921, andthen the unnecessary zwf and edd genes were deleted by inside out PCR.The upstream and downstream tdc sequences were obtained by PCR fromKJ122 chromosomal DNA as template. The P₂₆ promoter was obtained frombacteriophage SP01. The sequence of pAC19 is given as SEQ ID #1.

All constructions were done while growing strains on LB medium (10 gramsBacto-tryptone, 5 grams Bacto-yeast extract, and 5 grams sodiumchloride) supplemented as appropriate with antibiotic or sucrose. Toconstruct strain AC15, the cassette containing the artificial operon ofpAC19 was integrated into the chromosome of strain WG53, using a twostep gene replacement method previously described The cat, sacB cassettefor the first step was contained on plasmid pAC21, SEQ ID #2. pAC21 issimilar to pAC19, except that the artificial operon is replaced with acat, sacB cassette that contains a chloramphenicol resistance gene and acounterselectable sacB gene encoding levan sucrase The transforming DNAwas obtained by PCR form pAC21 for the first step and by PCR from pAC19for the second step.

Strain WG53 was obtained by deleting the ptsH, ptsI, and galP genes fromsuccinate producing strain KJ122, using a two step gene replacementmethod similar to that described in the above paragraph. The DNAsequence spanning the ptsHI deletion is given as SEQ ID #3. Note thatthis deletion leaves the crr gene intact, as well as native promotersthat naturally exist upstream from the ptsH gene. The DNA sequencespanning the galP deletion is given as SEQ ID #4.

While intermediate strain WG53 grew extremely poorly on minimal glucosemedium, strains KJ122 and AC15 grew well on minimal glucose medium,demonstrating that 1) the ptsHI and galP genes had been successfullydeleted in WG53, and 2) the glf, glk cassette was functional in AC15allowing glucose to be imported.

Example 2 Strain AC15 Produces Succinate as Well as Parent KJ122

Strains KJ122 and AC15 were grown under microaerobic condition in 7liter fermentors (New Brunswick Scientific) at 39° C. using a minimalmedium with glucose fed batch system. The starting volume of 3 literscontained potassium phosphate monobasic (18 mM), magnesium sulfate (2mM), betaine (1.33 mM), trace elements, Antifoam 204 (8 ppm) and 25 g/lglucose. The pH was adjusted initially to pH 7.0 and thereafter wasmaintained at pH 6.5 as acid was produced by addition of the ammoniumhydroxide/ammonium bicarbonate solution described below. The 150 mlinocula were grown aerobically and contained a minimal medium similar tothe above described medium, except that glucose was at 20 g/l andcalcium chloride was added to a final concentration of 0.1 mM. Agitationwas set at 750 RPM (revolutions per minute). When glucose decreased to 5g/l, a 650 g/l glucose feed was started and maintained at a rate aimedto keep the glucose concentration at about 5 g/l or less. The stocksolution used for neutralization contained both ammonium hydroxide andammonium bicarbonate (7 N NH₄OH and 3M NH₄HCO₃). AC15 was aerated at 35ml/min, while KJ122 was not given air other than what was present in thehead space, which was equilibrated with the atmosphere through abreathable sterile membrane filter. These were conditions that had beenshown to work well for each strain. Sugars, succinate, and byproductsfrom 48 hour samples were assayed by HPLC. The results of averagedduplicates are shown in Table 1. AC15 produced about the same titer asparent KJ122, but the acetate byproduct was significantly lower, and theyield on glucose was higher for AC15.

Example 3 Spontaneous “Red Mutants” Derived from AC15

KJ122 is able to ferment lactose, as evidenced by formation of redcolonies on MacConkey lactose plates (Beckton-Dickinson, Franklin Lakes,N.J.). However, AC15 does not ferment lactose, as evidence by producing“white” (beige) colonies on MacConkey lactose plates. This white colonyphenotype of AC15 results from binding and inhibition of lactosepermease (LacY) by unphosphorylated EIIA^(Glc) protein. This whitecolony phenotype is present in all strains deleted for ptsHI, since theenzymes required to phosphorylate EIIA^(Glc) are absent, and as aresult, all EIIA^(Glc) present in the cells remains unphosphorylated.Thus, ironically, E. coli ptsHI mutants are phenotypically Lac⁻, eventhough lactose is not imported by the PTS system in E. coli.

The inventors noticed by chance that when MacConkey lactose plates werestreaked with AC15 and allowed to incubate overnight at 37° C., and thenfor an extra day at room temperature (about 22° C.), a large number ofred colonies emerged from the lawn of white colonies that had grown overthe denser part of the streak. Upon restreaking of several of the redcolonies, it was observed that two classes of red colonies had evolved.We shall call the first class “solid red”, since the individual colonieswere uniformly red across the entire colony. A second class shall becalled “fried egg red”, since the individual colonies were red in thecenter, but the outer portion of the colonies were white or beige. Weshall call the strains giving rise to all types of red colonies onMacConkey lactose collectively “red mutants”.

A white colony of AC15, and four red mutants, named AC15-R1, -R2, -R3,and -R4 (two of which are solid red and two of which are fried egg red),were tested for succinate production in 500 ml microaerobic fermentors(Fleakers, Corning Glass, Corning, N.Y.) using a medium and methodsimilar to those described above for the 7 liter fermentors, with thedifferences being that the starting volume of the minimal medium was 200ml, the glucose was all batched in the starting medium at 100 g/l, noglucose was fed, agitation was with a magnetic stirring bar at 350 RPM,and no air was deliberately introduced or removed. The results are shownin Table 2. The two fried egg mutants performed similarly to parentAC15, while the two solid red mutants performed significantly worse thanparent AC15.

Genome sequencing of the parent AC15 and the four red mutants, using theIllumina HiSeq2000 system, revealed that both solid red mutants hadacquired one mutation each, and both of these mutations were in the crrgene, which encodes EIIA^(Glc). Both were judged to be null mutations.Both fried egg red mutants had acquired one mutation each, and both ofthese mutations were in the lactose operon. Both of these were judged tobe mutations that would lead to a higher level of expression of thelactose operon (one was a mutation in the lacO operator, and the otherwas a frameshift in lacI, the gene that encodes the Lac repressor. Allfour mutations made sense in that they could explain the observedphenotype of increased ability to ferment lactose. The crr nullmutations relieved the inhibition of the LacY permease, as would beexpected, while the lactose operon mutations would be expected tooverproduce LacY, allowing at least some escape from the inhibition.However, the crr null mutations clearly had an additional pleiotropiceffect, causing a decrease in the cells' ability to produce succinateunder our fermentation conditions. This was an unexpected effect thatwas not predicted.

Example 4

The Zymomonas mobilis glk gene is not essential for functioning of theglf gene in E. coli (084) Plasmid pSS2 was constructed using methodssimilar to those described above for pAC19. The only differences betweenpSS2 and pAC19 is that the, Z. mobilis glk gene was deleted from theartificial operon. In other aspects, such as vector backbone, thepromoter driving expression of glf, embedding the artificial operon inthe tdc flanking sequences, and orientation of the various components,pSS2 is similar to pAC19. The DNA sequence of pSS2 is given as SEQ ID#5.

The artificial operon from pSS2 was integrated at the tdc locus of KJ122as described above for the operon from pAC19, using the two step genereplacement method. Two isolates, which are presumably identical to eachother were named SS8-9 and SS8-11. These two new strains were comparedto AC15 in 500 ml microaerobic fermentors as described above in Example3. The results, which are averages of duplicate fermentors assayed at 48hours, are shown in Table 3. SS8-9 and SS8-11 both gave growth andsuccinate titers similar to that of AC15, while the acetate productionof both SS8 isolates was somewhat lower than that of AC15. Thus, the Z.mobilis glk gene is unnecessary for functioning of the glf gene in thiscontext, and the Z. mobilis glk gene might even be slightly harmful tothe fermentation parameters. Presumably, the SS8 isolates are using theendogenous E. coli glk gene to phosphorylate glucose.

Example 5 Metabolic Evolution of Strain AC15

As noted above in Example 3, strain AC15 preferred to receive a higherlevel of aeration than parent KJ122 in 7 liter fermentors. In order toobtain a derivative of AC15 that could thrive on less air, AC15 wassubjected to metabolic evolution in 500 ml fermentors with a startingvolume of 200 ml and no deliberate supply of aeration. The conditionswere microaerobic, since no measures were taken to remove oxygen. Asmall amount of air was assumed to leak into the fermentation vesselsduring the course of the evolution. The conditions for growth were asdescribed in Example 3. After 48 hours of growth, the culture wasdiluted 1:100 into a fresh fermentor containing 200 ml of fresh medium,and this step was then repeated 40 more times. Each one of theseinoculations to fresh medium shall be called a “transfer”. Thus, thestrain was subjected to a total of 41 transfers to fresh medium. Eachtransfer corresponds to about 7 generations of cell division. A sampleof the liquid culture from the last transfer was plated on a MacConkeylactose agar petri plate, and a single white colony was chosen and namedYSS41.

By varying the rate of aeration in 7 liter fermentors, it was determinedthat YSS41 performed well for succinate production with 5 ml/min of air,which was substantially less than the 35 ml/min required for optimalperformance of the parent AC15. With 5 ml/min air flow, YSS41 produced94 g/l succinate and 1.3 g/l acetate, for a succinate yield of 0.95 g/gglucose in 48 hours in a 7 liter fermentor.

YSS41 was compared to KJ122 for succinate production in a 20 literfermentor. The fermentation protocol was similar to that described abovefor 7 liter fermentors, except that the starting volume was 9 liters,and the aeration rate was 25 ml/min for both strains, conditions thathad been determined to be productive for both strains. The results for48 hour samples are shown in Table 4. The succinate titer for YSS41 was100 g/l (significantly higher than for KJ122), the acetate as 2.2 g/l(significantly lower than for KJ122), and the succinate yield was 0.95g/g glucose (a little lower than for KJ122). Thus, the evolved strainYSS41 was able to perform well in a 20 liter fermentor with an aerationrequirement that was no higher than for the ancestor strain KJ122.

Example 6 Stabilizing YSS41 Against Mutations in the Crr Gene

When streaked on MacConkey lactose plates, YSS41 still gave rise to redmutants, both of the solid red type and of the fried egg red type. Thecrr gene was sequenced for one isolate of each type. Strain MYR222, afried egg type had a wild type crr gene sequence. MYR223, a solid redtype, had an insertion element inserted in the crr open reading frame.The DNA sequence of the insertion element matched that of IS186. Thus,the pattern established for AC15 red mutants appeared to apply also toYSS41 red mutants. In 500 ml microaerobic fermentors, grown as inExample 4, MYR222 performed similarly to YSS41, while MYR223 performedmore poorly (see Table 5). Thus the potential loss of performance due toaccumulation of solid red mutants in a population remained a possibilitywith strain YSS41.

In order to solve this potential loss, a second copy of the crr gene wasintegrated into a site distant form the native crr locus. The crr gene,together with its flanking promoters and terminator were amplified byPCR using YSS41 chromosomal DNA as a template, and primers BY249 (SEQ ID#6) and BY250 (SEQ ID #7). The resulting blunt fragment was then ligatedinto a low copy plasmid derived from pCL1921 that contained a clone of aportion of the pflDC region from E. coli C at the unique BstZ171restriction site in the pflD open reading frame. The pflDC genes arehomologous to the pflBA genes that encode pyruvate-formate lyase and thepyruvate-formate lyase activating enzyme. The pflDC genes are notessential for E. coli, and deletion of either pflD or pflC has nosignificant effect on growth, so it was reasoned that insertion of acassette at that locus would not have any negative consequence forgrowth or succinate production. The resulting low copy plasmid, pMH68,contains the crr gene from YSS41 embedded in flanking sequences frompflDC, in a low copy plasmid. The DNA sequence of pMH68 is given as SEQID #8.

The integration cassette from pMH68 was amplified by PCR using primersBY124 (SEQ ID #9) and BY125 (SEQ ID #10), which were the same primersused to clone the pflDC genes to begin with. The integration cassettewas then integrated into the chromosome of YSS41, using the two stepgene replacement method. The resulting strain was named MH141, which isnow a merodiploid for crr, meaning that it contains two copies of a wildtype crr gene in two distant locations on the chromosome, one at itsnative locus, and the second inserted in the pflD open reading frame.

As expected, strain MH141 produced white colonies on MacConkey lactoseplates. If a heavy streak is made, and the plates are and allowed toincubate overnight at 37° C., and then for an extra day at roomtemperature, red colonies emerged from the lawn of white colonies thathad grown over the denser part of the streak. However, the number of redmutants arising from MH141 was significantly lower than for a similarstreak of YSS41 made on the same plate. 23 red mutants were picked fromYSS41 and 12 red mutants were picked from MH141, and all were restreakedon MacConkey lactose plates. When scored for the type of red mutant, 12of the 23 YSS41 red mutants were of the solid red type, while the other11 of the 23 were of the fried egg type. In contrast, all 12 of theMH141 red mutants were of the fried egg type. Thus, by duplicating thecrr gene in the chromosome, the rate of formation of the solid redmutants has been decreased by at least a factor of ten. One fried eggred mutant isolated from MH141 was named MH141-R1 and tested in 500 mlmicroaerobic fermentors as described above (see Table 5). Both MH141 andMH141-R1 performed similarly to parent YSS41 with respect to growth,succinate titer, and acetate titer. Thus, a more stable strain, MH141,has been constructed that uses facilitated diffusion for glucose import,and which produces a higher titer of succinate and a lower titer of thebyproduct acetate when compared to the ancestor strain KJ122, which usesa the GalP system for glucose import.

Example 7 YSS41 Acquired Mutations in the Glf, Glk Cassette DuringMetabolic Evolution

The DNA sequences of the glf, glk expression cassettes in AC15 and YSS41were determined. The regions were amplified by PCR and the resultingfragments were sequenced over the glf and glk genes and more than 200base pairs upstream and downstream, by the dideoxy chain terminationmethod. The sequenced region corresponds to bases 4976 to 7920 of pAC19,given in SEQ ID #1. Two mutations were found that were acquired duringthe evolution of YSS41. The first mutation was a G to A change at basenumber 7742 of SEQ ID #1. This base is in the 5′ untranslated region ofthe glf, glk mRNA transcript, just upstream from the glf open readingframe, and results in a C to U change at base −22 relative to the ATGstart codon, or +15 relative to the start of transcription, in the glfmRNA (messenger RNA). This mutation is expected to increase or decreasethe rate of translation of the glf open reading frame. The secondmutation was a G to A change at base number 6173 of SEQ ID #1. This baseis in the 5′ untranslated region just upstream from the glk open readingframe, and results in a C to U change at base −15 relative to the ATGstart codon in the glk mRNA. This mutation is expected to increase ordecrease the rate of translation of the glk open reading frame. Thus,the evolution of YSS41 resulted in a more optimal balance of expressionbetween the glf and glk open reading frames, to result in a strain thatoutgrew and outperformed the parent strain AC15.

Other mutations that alter the rate of transcription or expression ofthe glf and glk genes, or that alter the concentration, specificactivity, or stability of the glf and glk proteins, can similarlyachieve a more optimal balance between the two encoded proteins willalso benefit growth and production of a desired chemical. These otheralternative mutations can be obtained by the using the method describedabove for YSS41. This method can also be applied to strains engineeredto produce products other than succinate, where the ability to usefacilitated diffusion or sugar import has been engineered into thestrain.

Example 8

Fermentation of KJ122 and YSS41 after optimization of air flow rate forYSS41 (095) The optimum air flow rate for parent strain KJ122 had beendetermined to be 25 ml/minute in a 20 liter fermentor. At the air flowrate of 25 ml/min, YSS41 strain showed better succinate titer and yieldwhen compared to that of KJ122. Further improvement in succinate yieldand titer with YSS41 strain was obtained by increasing the air flow rateto 50 ml/min. Thus the optimal air flow rate for YSS41 strain withreference to succinate yield and titer seems to be different from thatof KJ122. Table 6 provides fermentation results in the 20 literfermentor under the optimized air flow conditions for each strain. YSS41outperformed parent KJ122 in titer, yield, and acetate byproductformation. The initial volume of the fermentation was 9500 ml. Afterfeeding glucose and neutralizing with base the final volume was 12500ml.

REFERENCES

All references are listed for the convenience of the reader. Eachreference is incorporated by reference in its entirety.

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TABLE 1 Production of Succinate by AC15 in 7 Liter Fermentors RelevantAeration Succinate Acetate Yield g/g Strain genotype ml/min g/l g/lglucose KJ122 parent, ptsI*, 0 87 5.2 0.83 galP+ AC15 KJ122, ΔptsHI, 3587 2.7 0.88 ΔgalP, P₂₆-glf, glk

TABLE 2 Production of succinate by AC15 red mutants in 500 mlmicroaerobic fermentors Colony phenotype on MacConkey Succinate AcetateMutation Strain lactose g/l g/l OD₆₀₀ found AC15 white 74 2.4 7.5 noneAC15-R1 solid red 51 9.0 6.5 crr Lys16 frameshift AC15-R3 solid red 656.6 7.0 crr Met1Ile AC15-R2 fried egg red 73 2.4 8.0 lacO G11A AC15-R4fried egg red 73 3.0 7.5 lacI Asp300 frameshift

TABLE 3 Succinate production by SS8 isolates in 500 ml microaerobicfermentors Succinate Acetate Strain Relevant genotype g/l g/l OD₆₀₀ AC15KJ122, ΔptsHI, ΔgalP, 64 6.0 7.5 P₂₆-glf, glk SS8-9 KJ122, ΔptsHI,ΔgalP, 64 4.2 8.5 P₂₆-glf SS8-11 KJ122, ΔptsHI, ΔgalP, 64 3.6 8.2P₂₆-glf

TABLE 4 Succinic acid production by YSS-41, in a 20 liter fermentor Airflow Yield on Relevant rate Succinate Acetate glucose Strain genotypeml/min g/l g/l g/g KJ122 ptsI* 25 87 6.8 1.00 KJ122 ptsI* 25 85 6.8 0.98YSS41 KJ122□□ΔptsHI, 25 100. 2.2 0.95 ΔgalP, P₂₆-glf, glk, evolved

TABLE 5 Succinate production in 500 ml microaerobic fermentors by MH141,a merodiploid for crr+. Colony phenotype Relevant on MacConkey SuccinateAcetate Strain genotype lactose g/l g/l OD₆₀₀ YSS41 AC15, white 67 3.910.0 evolved, crr⁺ MH141 YSS41, white 68 3.2 8.5 ΔpflD::crr⁺

TABLE 6 Succinate production by E. coli strains KJ122 and YSS41 in 20liter fermentors under optimized aeration conditions for both strainsSuccinate Air flow Succinate yield on Acetate Cell rate titer glucosetiter mass as Strain (ml/min) (g/l) (g/g) (g/l) OD600 KJ122 25 81 0.863.9 12 YSS41 50 96 0.98 2.5 13 YSS41 25 93 0.98 2.5 13

What is claimed is:
 1. A bacterium producing more than 30 grams perliter of a desired chemical, wherein one of the biosyntheticintermediates for said desired chemical is phosphoenolpyruvate, and saidbacterium contains at least one exogenous gene that encodes a proteinthat functions in the facilitated diffusion of a sugar.
 2. The bacteriumof claim 1, wherein the bacterium is selected from a group consisting ofGluconobacter oxydans, Gluconobacter asaii, Achromobacter delmarvae,Achromobacter viscosus, Achromobacter lacticum, Agrobacteriumtumefaciens, Agrobacterium radiobacter, Alcaligenes faecalis,Arthrobacter citreus, Arthrobacter tumescens, Arthrobacter paraffineus,Arthrobacter hydrocarboglutamicus, Arthrobacter oxydans, Aureobacteriumsaperdae, Azotobacter indicus, Brevibacterium ammoniagenes, divaricatum,Brevibacterium lactofermentum, Brevibacterium flavum, Brevibacteriumglobosum, Brevibacterium fuscum, Brevibacterium ketoglutamicum,Brevibacterium helcolum, Brevibacterium pusillum, Brevibacteriumtestaceum, Brevibacterium roseum, Brevibacterium immariophilium,Brevibacterium linens, Brevibacterium protopharmiae, Corynebacteriumacetophilum, Corynebacterium glutamicum, Corynebacterium callunae,Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum,Enterobacter aerogenes, Erwinia amylovora, Erwinia carotovora, Erwiniaherbicola, Erwinia chrysanthemi, Flavobacterium peregrinum,Flavobacterium fucatum, Flavobacterium aurantinum, Flavobacteriumrhenanum, Flavobacterium sewanense, Flavobacterium breve, Flavobacteriummeningosepticum, Micrococcus sp. CCM825, Morganella morganii, Nocardiaopaca, Nocardia rugosa, Planococcus eucinatus, Proteus rettgeri,Propionibacterium shermanii, Pseudomonas synxantha, Pseudomonasazotoformans, Pseudomonas fluorescens, Pseudomonas ovalis, Pseudomonasstutzeri, Pseudomonas acidovolans, Pseudomonas mucidolens, Pseudomonastestosteroni, Pseudomonas aeruginosa, Rhodococcus erythropolis,Rhodococcus rhodochrous, Rhodococcus sp. ATCC 15592, Rhodococcus sp.ATCC 19070, Sporosarcina ureae, Staphylococcus aureus, Vibriometschnikovii, Vibrio tyrogenes, Actinomadura madurae, Actinomycesviolaceochromogenes, Kitasatosporia parulosa, Streptomyces coelicolor,Streptomyces flavelus, Streptomyces griseolus, Streptomyces lividans,Streptomyces olivaceus, Streptomyces tanashiensis, Streptomycesvirginiae, Streptomyces antibioticus, Streptomyces cacaoi, Streptomyceslavendulae, Streptomyces viridochromogenes, Aeromonas salmonicida,Bacillus pumilus, Bacillus circulans, Bacillus thiaminolyticus,Escherichia freundii, Microbacterium ammoniaphilum, Serratia marcescens,Salmonella typhimurium, Salmonella schottmulleri, Bacillus subtilis,Bacillus licheniformis, Bacillus amylolliquefaciens and Xanthomonascitri.
 3. The bacterium of claim 1, wherein the bacterium is selectedfrom a group consisting of Escherichia coli, Corynebacterium glutamicumBrevibacteium flavum, Mannhemia succiniproducens and Anaerobiospirilumsucciniproducens.
 4. The bacterium according to claim 1, wherein saiddesired chemical is selected from a group consisting of succinic acid,fumaric acid, glucaric acid, malonic acid, maleic acid, 2,5-furandicarboxylic acid, propionic acid, 3-hydroxypropionic acid, asparticacid, glucaric acid, glutamic acid, itaconic acid, levulinic acid,3-hydroxybutryolactone, and butanediols such as 1,4 butnaediol,1,3-butanediol and 2,3-butanediol.
 5. The bacterium according to claim1, wherein said desired chemical is succinate.
 6. The bacteriumaccording to claim 1, wherein said desired chemical is cis, cis-muconicacid.
 7. The bacterium according to claim 1, wherein said desiredchemical is an aromatic biochemical.
 8. A bacterium according to claim1, wherein the bacterium is a PTS⁻ bacterial strain.
 9. A bacteriumaccording to claim 1, wherein the bacterium has a reduced level ofphosphotransferase activity compared to a related wild type strain. 10.A bacterium of claim 1 further comprising a mutation or deletion in oneor more genes that encode proteins that functions in aphosphotransferase system for sugar import, said mutated or deleted genebeing other than a crr gene.
 11. A Bacterium of claim 10 furthercomprising a deletion in a gene that encodes a sugar importer thatfunctions using proton symport.
 12. A bacterium of claim 10 wherein saidmutated or deleted gene is a ptsH gene or a homolog thereof.
 13. Abacterium of claim 10 wherein said mutated or deleted gene ptsI gene ora homolog thereof.
 14. A bacterium of claim 10 wherein said mutated ordeleted genes are selected from a group consisting of ptsH, ptsI,homolog of ptsH and homolog of ptsI.
 15. A bacterium according to claim1, wherein the bacterium is a galP⁻ bacterial stain.
 16. The bacteriumof claim 1, wherein said exogenous gene is contained on a replicatingplasmid.
 17. The bacterium of claim 1, wherein said exogenous gene isintegrated into the host chromosome.
 18. The bacterium of claim 1wherein said exogenous gene is a glf gene.
 19. The bacterium of claim 1wherein said exogenous genes are a glf and a glk genes.
 20. Thebacterium of claim 1 in which said exogenous genes are a glf gene and afrk gene.
 21. The bacterium of claim 1 in which said exogenous gene isderived from a yeast.
 22. The bacterium of claim 1 in which saidexogenous gene or genes are derived from a strain of Zymomonas mobilis.23. A bacterium of claim 1, wherein said bacterium is grown in minimalmedium.
 24. A bacterium of claim 1 wherein said bacterium produces morethan 64 grams per liter of a desired chemical.
 25. A bacterium of claim1 wherein said bacterium produces more than 83 grams per liter of adesired chemical.
 26. A bacterium containing two or more copies of afunctional crr gene or a functional homolog of a crr gene.
 27. A methodfor producing a desired chemical comprising the steps of: growing abacterium of claim 1 in a minimal fermentation medium; and optionallypurifying said chemical from the fermentation medium.
 28. A method forproducing succinic acid comprising the steps of: growing a bacteriumcomprising at least one exogenous gene that encodes a protein thatfunction in the facilitated diffusion of sugar and producing at least 60grams of succinate per liter and less than 4.2 grams of acetate perliter in a minimal fermentation medium; and optionally purifyingsuccinic acid from the fermentation medium.
 29. A method for improvingthe titer and yield of a desired chemical produced by a bacterial strainthat has been engineered to use facilitated diffusion for import of asugar, comprising the steps of: subjecting a parent strain to serialtransfers into fresh liquid medium; plating the resulting culture forsingle colonies on a petri plate; and choosing a single colony.
 30. Abacterial strain engineered to use facilitated diffusion for import of asugar comprising a glf-glk operon, wherein said bacterial strain hasbeen evolved for improved titer and yield for succinate production, andfurther comprising one or more mutations in the glf-glk operon.
 31. Abacterial strain of claim 30 in which said one or more mutations alterbases in the DNA sequence that corresponds to the 5′ untranslated leaderregion of an mRNA that encodes either of said glf or glk gene.
 32. Abacterial strain engineered to use facilitated diffusion for import of asugar comprising a glf gene, wherein said bacterial strain has beenevolved for improved titer and yield for succinate production, and saidglf gene comprises one or more mutations.
 33. A bacterial strainengineered to use facilitated diffusion for import of a sugar comprisinga glk gene, wherein said bacterial strain has been evolved for improvedtiter and yield for succinate production, and said glk gene comprisesone or more mutations.